Decades of research on the myriad of biological activities that the polyamines, putrescine, spermidine and spermine play in cellular processes have shown the profound role they play in life (Cohen, S. S., “A Guide to the Polyamines” 1998, Oxford University Press, New York). As polycations at physiological pH, they bind tightly to and strongly modulate the biological activities of all of the anionic cellular components. Specific and strong interactions have been associated with DNA and RNA together with their associated chromatin proteins (Tabor, H. et al. 1,4-Diaminobutrane (putrescine), spermidine, and spermine. Ann Rev. Biochem. 1976, 45, 285–306; Matthews, H. R. Polyamines, chromatin structure and transcription. BioEssays, 1993, 15, 561–566). Spermine has been shown to function directly as a free radical scavenger that protects DNA from insults by reactive oxygen species (Ha, H. C. et al. Proc. Natl. Acad. Sci. USA, 1998, 95, 11140–11145). Specific interactions of multicationic polyamines with microtubules has been recently shown (Wolff, J. Promotion of Microtubule Assembly by Oligocations: Cooperativity between Charged Groups. Biochemistry, 1998, 37, 10722–10729; Webb, H. K. et al., J. Med. Chem 1999, in press). Allosteric regulation of membrane-bound enzymes including acetylcholinesterase has been shown (Kossorotow, A. et al. Regulatory effects of polyamines on membrane-bound acetylcholinesterase. Biochem. J. 1974, 144, 21–27). Polyamines have a direct influence on many neurotransmitter receptors and ion channels (Carter, C. The Neuropharmacology of Polyamines, 1994, Academic Press, San Diego, Calif.; Williams, K. Interaction of polyamines with ion channels, Biochem. J., 1997, 325, 289–297). Specific polyamine binding sites have also been demonstrated for the NMDA receptor complex (Ransom, R. W. et al. Cooperative modulation of [3H]MK-801 Binding to the N-Methyl-D-Aspartate Receptor-Ion Channel Complex by L-Glutamate, Glycine, and Polyamines. J. Neurochem. 1988, 51, 830–836; Williams, K. et al. Minireview: Modulation of the NMDA receptor by polyamines. Life Sci 1991, 48, 469–498).
Many stimuli involved in both normal and neoplastic growth activate the polyamine biosynthetic pathway. A great number of multidisciplinary studies have shown that the intracellular concentrations of the polyamines is highly regulated at many steps in their biosynthesis, catabolism and transport. The fact that cells contain such complex apparatus for the tight control of the levels of these molecules shows that only a very narrow concentration range is tolerated. Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, catalyzes the production of putrescine from its precursor ornithine. This enzyme, with a very short biological half-life, is one of the most inducible mammalian enzymes known (Russell, D. et al. Amine synthesis in rapidly growing tissues: ornithine decarboxylase activity in regenerating rat liver, chick embryo, and various tumors. Proc. Natl. Acad. Sci. USA 1968, 60, 1420–1427). Many biological stimuli involved in cellular growth have been shown to induce this enzyme and a distinct growth advantage is gained by induction of ODC (Alhonen-Hongisto, L. et al. Tumourigenicity, cell-surface glycoprotein changes and ornithine decarboxylase gene pattern in Ehrlich ascites-carcinoma cells. Biochem. J. 1985, 229, 711–715). An increase in the activity of ODC has been associated with tumor growth (Jänne, J. et al. Polyamines in rapid growth and cancer. Biochim. Biophys. Acta 1978, 473, 241–493; Scalabrino, G. et al. Polyamines in mammalian tumors. Part I. Adv. Cancer Res. 1981, 35, 151–268; Scalabrino, G. et al. Polyamines in mammalian tumors. Part II. Adv. Cancer Res. 1982, 36, 1–102). Feedback inhibition of ODC activity is mediated by ODC-antizyme protein. Following elevation of polyamine concentrations, a polyamine-stimulated +1 frameshift of the ODC-antizyme mRNA reading frame causes elevation of this ODC-inhibiting protein (Hayashi, S. et al. Ornithine decarboxylase antizyme: a novel type of regulatory protein. TIBS, 1996, 21, 27–30; Matsufuji, S. et al. EMBO Journal, 1996, 15, 1360–1370). The ODC-antizyme protein binds to ODC with high affinity to form an inactive complex that is then tagged for degradation in an ATP-dependent fashion by the 26S proteosome (Heller, J. S. et al. Proc. Natl. Aced. Sci. USA 1976, 73,1858–1862; Murakami, Y. et al. Ornithine decarboxylase is degraded by the 26S proteosome without ubiquitination. Nature, 1992, 360, 597–599). ODC-antizyme also represses the polyamine uptake system of cells (Suzuki, T. et al. Antizyme protects against abnormal accumulation and toxicity of polyamines in ornithine decarboxylase-overproducing cells. Proc. Natl. Acad. Sci. USA. 1994, 91, 8930–8934).
The polyamine catabolism pathway is important to prevent the toxic effects of excess polyamines on cells (Seiler, N. Functions of polyamine acetylation. Can. J. Physiol. Pharmacol. 1987, 65, 2024–2035; Seiler, N. Polyamine oxidase, properties and functions. Progress in Brain Res. 1995, 106, 333–344). This pathway is used by the cell to interconvert the various polyamines and to eliminate excess polyamines before they reach toxic levels. This pathway introduces no additional carbon precursors into the polyamine pool.
Polyamine transport into mammalian cells is energy and temperature dependent, saturable, carrier mediated and operates against a substantial concentration gradient (Seiler, N. et al. Polyamine transport in mammalian cells. Int. J. Biochem 1990, 22, 211–218; Khan, N. A.; Quemener, V. et al. Characterization of polyamine transport pathways, in Neuropharmacology of Polyamines (Carter, C., ed.), 1994, Academic, San Diego, pp. 37–60). Ample experimental proof exists that polyamine concentration homeostasis is mediated via this transport system. Changes in the requirements for polyamines in response to growth stimulation is reflected by increases in the transport activity. Stimulation of human fibroblasts to cell proliferation by serum or epidermal growth factor was followed by an 18–100 fold increase in the uptake of putrescine (DiPasquale, A. et al. Epidermal growth factor stimulates putrescine transport and ornithine decarboxylase activity in cultures human fibroblasts. Exp. Cell Res. 1978, 116, 317–323; Pohjanpelto, P. Putrescine transport is greatly increased in human fibroblasts initiated to proliferate J. Cell Biol. 1976, 68, 512–520). Tumors have been shown to have an increased rate of putrescine uptake (Volkow, N. et al. Labeled putrescine as a probe in brain tumors. Science, 1983, 221, 673–675; Moulinoux, J- P. et al. Biological significance of circulating polyamines in oncology. Cell. Mol. Biol. 1991, 37, 773–783). Inhibition of polyamine biosynthesis in cells in culture by α-difluoromethylornithine (DFMO), a well-studied mechanism-based inhibitor of ODC, causes a substantial depletion of intracellular putrescine and spermidine with resultant cell growth inhibition. Upon supplementing the culture media with exogenous polyamines this depletion causes transport activity to rise several-fold (Bogle, R. G. et al. Endothelial polyamine uptake: selective stimulation by L-arginine deprivation or polyamine depletion. Am. J. Physiol. 1994, 266, C776–C783; Alhonen-Hongisto, L. et al. Intracellular putrescine deprivation induces uptake of the natural polyamines and methylglyoxal bis(guanylhydrazone). Biochem. J. 1980, 192, 941–945). The cells then returned to their original rate of growth.
Several experimental lines of evidence support the conclusion that increased effectiveness of ODC inhibition can be obtained by interfering with the polyamine transport apparatus. A mutant L1210 leukemia cell line was shown to have greatly reduced polyamine transport activity following selection for resistance to methylglycoxal bis(guanylhydrazone) (MGBG), an extremely cytotoxic AdoMetDC inhibitor that is taken up by the same transport system as the polyamines. Mice inoculated with these cells had a much greater response to DFMO treatment (87% increase in median survival time; 13 of 40 mice cured) than mice inoculated with the parental cell line (22% increase in median survival time). See Persson, L. et al. Curative effect of d,1-2-difluoromethylornithine on mice bearing mutant L1210 leukemia cells deficient in polyamine uptake. Cancer Res 1988, 48, 4807–4811. A significant source of extracellular polyamines is produced by the microbial flora in the gastrointestinal tract (Sarhan, S. et al. The gastrointestinal tract as polyamine source for tumor growth. Anticancer Res 1989, 9, 215–224). When this source of polyamines is removed by decontamination of this flora, DFMO's previous moderate growth inhibitory effects on Lewis lung carcinoma cells or L1210 zenografts is markedly potentiated (Hessels, J. et al. Limitation of dietary polyamines and arginine and the gastrointestinal synthesis of putrescine potentiates the cytostatic effect of a-difluoromethylornithine in L1210 bearing mice. Int. Symp. Polyamines in Biochemical and Clinical Research, Sorrento (Italy), 1988, Abstr. P105). An additional source of polyamines is from dietary sources (Bardocz, S. et al. Polyamines in food; implications for growth and health. J. Biochem Nutr. 1993, 4, 66–71). By feeding a polyamine-free diet to DFMO-treated nude mice the MCF-7 human breast cancer zenografts contained greatly reduced levels of putrescine in comparison to DFMO treatment alone (Levêque, J. et al. The gastrointestinal polyamine source depletion enhances DFMO induced polyamine depletion in MCF-7 human breast cancer cells in vivo. Anticancer Res. 1998, 18, 2663–2668). In additional animal models, complete polyamine deprivation also enhanced DFMO's growth inhibitory effectiveness (Moulinoux, J. P. et al. Inhibition of growth of the U-251 human glioblastoma in nude mice by polyamine deprivation. Anticancer Res. 1991, 11, 175–180; Quemener, V. et al. Polyamine deprivation enhances antitumoral efficacy of chemotherapy. Anticancer Res. 1992, 12, 1447–1454; Chamaillard, L. et al. Polyamine deprivation prevents the development of tumour-induced immune suppression. Br. J. Cancer 1997, 76, 365–370).
The Polyamine Transporter (PATr)
The increased demand for polyamines by rapidly growing, transformed cancer cells is only partially met by an increased rate of synthesis. To exploit this increased need for polyamines, synthesis inhibitors have been sought. Additionally, lowering polyamine concentrations can result in aberrations in chromatin structure leading to cell death or inhibition of proliferation (Quemener, V. et al., Anticancer Res. 14:443448, 1994; Porter, C. W. et al., Cancer Res. 53:581–586, 1993). It has become increasingly apparent that the initial disappointing results observed in the clinic with polyamine synthesis inhibitors arises from compensatory increases in transport of polyamines by a specific active transport system (Seiler, N. et al., Int. J. Biochem 22:211–218, 1990; Seiler, N. et al, J. Biochem. Cell Biol. 28:843–861, 1996). The promising results observed in cell culture with a suicide substrate inhibitor of ornithine decarboxylase, α-difluoromethylornithine (DFMO), or with an inhibitor of S-adenosylmethionine decarboxylase, methylglyoxal bis(guanylhydrazone) (MGBG) did not transfer to human clinical trials (Schecter, P. J. et al., In Inhibition of Polyamine Metabolism. Biological Significance and Basis for New Therapies; McCann, P. P. et al., eds; 1987, pp 345–364). Since the only two avenues for carbon transfer into polyamine pools are synthesis or transport, simultaneous inhibition of both of these pathways is considered by the present inventors to be a promising anticancer therapeutic approach.
A study confirming the validity of this chemotherapeutic approach used transplanted murine L1210 leukemia cells that were deficient in PAT. Mice transplanted with the wild-type L1210 cancer cells (with intact PAT) died after 12 days, even when treated with DFMO. In contrast, DFMO mice transplanted with PAT-deficient L1210 cells lived longer than 60 days (Ask, A. et al., Cancer Lett. 66:29–34, 1992). These authors also showed that treatment of mice harboring wild-type L1210 cells with a combination of (1) DFMO (2) a low polyamine diet and (3) antibiotics (which decrease polyamine production by gut flora) resulted in prolonged survival compared to treatment with DFMO alone.
Augmented PAT into cancer cells promotes cell killing. J. L. Holley et al. (Cancer Res. 52:4190–4195, 1992) showed up to a 225-fold increase in cytotoxicity of a chlorambucil-spermidine conjugate compared to chlorambucil alone. A series of nitroimidazole-polyamine conjugates were also effective (Holley, J. L. et al., Biochem. Pharmacol. 43:763–769, 1992). Others showed that mice infected with a multi-drug resistant strain of malaria were cured by treatment with a chloroquinoline-putrescine conjugate (Singh, S. et al., J. Biol. Chem. 272:13506–13511, 1997). Thus, the effectiveness of cytotoxic compounds could be enhanced by their conjugation with polyamines. These effects may have been due to the exploitation of the PAT system to deliver these compounds into cancer cells.
The gene for the polyamine transport protein has been cloned from Escherichia coli and recently from yeast (Kashiwagi, K. et al. J. Biol. Chem. 1990, 265, 20893–20897; Tomitori, H. et al. Identification of a gene for a polyamine transport protein in yeast. J. Biol. Chem. 1999, 274, 3265–3267). The genes for the mammalian transporter await identification. The transporter from E. coli has been crystallized and its X-ray structure has been determined (Sugiyama, S. et al. Crystal structure of PotD, the primary receptor of the polyamine transport system in Escherichia coli. J. Biol. Chem. 1996, 271, 9519–9525). This structure represents one of only a few but growing number determined for spermidine-binding proteins. Since this structure was determined on a prokaryotic species its use in the design of mammalian transport inhibitors was deemed to be of limited value. Despite this, several insights were obtained and used through analysis of this structure. In addition to the expected presence of carboxylate residues positioned to form salt bridges with the protonated amino groups of spermidine, numerous aromatic residues, especially tryptophan residues appeared to strengthen hydrophobic interactions with the methylene groups of the substrate. Additionally, a H2O molecule was positioned at one end of spermidine substrate, providing stronger interactions with the ionic residues in this position.
Several researchers have studied the ability of polyamine analogs to inhibit the uptake of 3H-spermidine into cells. Bergeron and coworkers studied the effect of addition of different alkyl group substitution on the terminal nitrogen atoms of spermidine or spermine analogs (Bergeron, R. J. et al. Antiproliferative properties of polyamine analogues: a structure-activity study. J. Med. Chem. 1994, 37, 3464–3476). They showed larger alkyl groups diminished the ability to prevent uptake of radiolabeled spermidine. They later concluded that increases in the number of methylenes between the nitrogen atoms decreased the ability to compete for 3H spermidine uptake (Bergeron, R. J. et al. A comparison of structure-activity relationships between spermidine and spermine antineoplastics J. Med. Chem. 1997, 40, 1475–1494). Of greater importance to the present work was their conclusion that the polyamine transport apparatus requires only three cationic centers for polyamine recognition and transport (Porter, C. W. et al. J. Cancer Res. 1984, 44, 126–128). Two groups analyzed literature examples of the polyamine analogs ability to inhibit 3H spermidine uptake into L1210 cells by CoMFA and QSAR methods (Li, Y. et al. Comparative Molecular field analysis-based predictive model of structure-function relationships of polyamine transport inhibitors in L1210 cells. Cancer Res-1997, 57, 234–239; Xia, C. Q. et al. QSAR analysis of polyamine transport inhibitors in L1210 cells. J. Drug Target 1998, 6, 65–77).
Polyamine Transport (PAT) Assays
There is no known high-throughput assay for measuring PAT. A radiochemical assay is used for biochemical analysis of transport and has been used to study PAT in yeast and a variety of mammalian cells (Kakinuma, Y. et al., Biochem. Biophys. Res. Comm. 216:985–992, 1995; Seiler, N. et al., Int. J. Biochem. Cell Biol 28:843–861, 1996). See, for example Huber, M. et al. Cancer Res. 55:934–943, 1995.
The radiometric assay uses radiolabeled polyamines such as putrescine, spermidine or spermine, but, due to the low signal, large numbers of adherent or non-adherent cells are required. Additional care is required with spermine due to its nonspecific adsorption to cells and plastics. Cells are mixed with the test compounds and the radiolabeled polyamine to initiate the assay. The cells are incubated for 1–60 minutes, depending on cell type. The assay is terminated by removal of the medium and cooling the plates to 4° C. The cells are then washed with cold medium three times, dissolved in 0.1% sodium dodecyl sulfate and the radioactivity in solution is then determined by scintillation counting. This assay is difficult to scale up to a high throughput procedure due to the low signal from the radiolabel and the handling requirements inherent in procedures with radioactivity.
A great number of polyamine amide natural products have been recently been discovered in the venom of arthropods such as spiders and wasps. These acylpolyamine analogs have been shown to have specific and strong interactions with the neuromuscular junctions of insects (Moya, E. et al. Syntheses and neuropharmacological properties of arthropod polyaminne amide toxins. Neuropharmacology of Polyamines (Carter, C., ed.), 1994, Academic, San Diego, pp. 167–184). With this capability these toxins give the insect predators the ability to paralyze or kill their prey. Most of these natural products have the common molecular features of a polyamine moiety (many with structurally diverse polyamine analogs) connected through an amide with an aromatic amino acid structural analog. Simpler synthetic analogs have been sought that attempt to maximize interactions with either crustacean neuromuscular synapses or mammalian glutamate receptors (Asami, T. et al. Acylpolyamines mimic the action of Joro spider toxin (JSTX) on crustacean muscle glutamate receptors. Biomedical Res. 1989, 10, 185–189; Raditsch, M. et al. Polyamine spider toxins and mammalian N-methyl-D-aspartate receptors. Structural basis for channel blocking and binding of argiotoxin 636. Eur. J. Biochem. 1996, 240, 416–426; Tsubokawa, H. et al. Effects of a spider toxin and its analoque on glutamate-activated currents in the nippocampal CA1 Neuron after ischemia. J. Neurophys. 1995, 74, 218–225).
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